Two lines of research were conducted to investigate the biosynthesis of pertussis toxin (PT) as well as the structure/function relationships of the toxins. The first line of research investigated the structure of the PT operon and the second involved the investigation of forms of PT encoded by the ptx genes of B. parapertussis and B. bronchiseptica.Previously, the ptx operon of B. pertussis was thought to comprise only the gens encoding the structural subunits of the toxin. Recently, we discovered a set of genes which encode accessory proteins essential for the secretion of pertussis toxin. These ptl genes were found to be located directly downstream from the ptx genes. The location of the promoter of the ptl genes was uncertain. We asked the question whether the ptl genes constituted an operon or whether they were part of the ptx operon and were therefore controlled by the ptx promoter. We constructed a strain of B. pertussis that lacked the ptx promoter but contained the remainder of the ptx-ptl region. We found that this strain did not produce PtlF as visualized by immunoblot techniques, suggesting that the Ptl bronchiseptica with that of B. pertussis. B. broncheseptica has both ptx and ptl genes but has a defective promoter. Previously, it has been shown that B. bronchiseptica produces neither PT nor Ptl proteins. The strain of B. broncheseptica which contained the B. pertussis ptx promoter did make PtlF. These results are consistent with the interpretation that the ptx operon comprises both ptx and ptl genes. This information will be useful for the design of vaccine strains which both produce and secrete large quantities of PT. While the current dogma in the field is that only B. pertussis produces PT, recent clinical trials have provided provocative results which may suggest otherwise. A number of clinical investigators have reported that sera from children infected with either B. parapertussis or B. bronchiseptica can show antibody rises to PT. It has been known for some time that both B. parapertussis and B. bronchiseptica contain ptx genes but the genes were thought to be transcriptionally silent. We asked the question of whether the ptx genes of B. parapertussis and B. bronchiseptica could produce functionally active PT, if the genes were expressed. We constructed strains of both B. parapertussis and B. bronchiseptica which had the B. pertussis ptx promoter inserted immediately in front of the ptx-ptl regions of those strains. We found that both species produced active PT as well as at lease certain of the Ptl proteins. The S2 subunit was confirmed to be prematurely terminated in B. parapertussis. Nonetheless, an active toxin was produced, although this toxins appeared to be more labile that PT. These results suggest that, if the ptx-ptl genes of B. parapertussis and B. bronchiseptica were expressed during the disease process, they would be functionally active and might contribute to pathogenesis.